Cell culture media and methods

ABSTRACT

Compositions and methods are described for preparing media, feeds, and supplements. Such methods and medias may display increased stability of labile components and may use, for example, microsuspension and/or encapsulation technologies, chelation, and optionally, coating and/or mixing the labile compounds with anti-oxidants. The compositions may withstand thermal and/or irradiation treatment and have reduced virus number. These techniques may result in product with extended shelf-life, extended release of their internal components into culture, or in product that can be added aseptically into a bioreactor using minimal volumes. The compositions and methods may optimize the bioproduction workflow and increase efficiency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/198,237, filed Jun. 30, 2016, which is a continuation of U.S.application Ser. No. 14/367,413, filed Jun. 20, 2014, now U.S. Pat. No.9,410,118, which is a National Stage filing of PCT/US2012/071411, filedDec. 21, 2012, which claims the benefit of U.S. Provisional ApplicationNo. 61/579,432, filed Dec. 22, 2011, under 35 U.S.C. § 119(e), all ofwhich disclosures are herein incorporated by reference in theirentirety.

TECHNICAL FIELD

Provided herein are compositions, methods and uses relating to noveltypes of cell culture media, feeds and supplements. The media, feeds andsupplements have several desirable properties, which include but are notlimited to, having certain components beyond their normal solubilitylimits, increased tolerance of radiation, extended release properties,high solubility, increased shelf life, and thermostability. Suchcharacteristics, in certain embodiments, improve customer workflows andbioreactor productivity.

BRIEF DESCRIPTION OF THE FIGURES

The following figures, which are described below and which areincorporated in and constitute a part of the specification, illustrateexemplary embodiments that are not to be considered limiting to thescope of the disclosure.

FIG. 1 shows a new procedure developed for making micro/nanosuspensionsof cell culture media, feed and/or supplement components.

FIG. 2 shows a comparison of the performance of cell growth, proteinproduction and delivery of microsuspensions and liquid feeds.Microsuspensions performed comparably to liquid feeds under theconditions tested and showed higher delivery efficacy than liquid feeds.

FIG. 3 a schematic representation of an encapsulated microsuspensionbead, which is additionally coated for extended release.

FIG. 4 shows the impact of PLGA coating on delaying the release ofcomponents from within the microencapsulated microsuspensionpreparations. a) top panel: no coating; b) bottom panel: comparison ofcoating with PLGA A and PLGA B. A and B have different ratios oflactide: glycolide. PLGA B with an 85:15 ratio showed good extendedrelease properties and maintained its shape an ability to releasecomponents >15 days in solution.

FIG. 5 shows the effect of irradiation on the preparations of thisdisclosure. Comparison of protein production efficiency in cellscultured with i) irradiated microsuspensions, ii) dry, encapsulatedmicrosuspensions are similar to those in iii) control, non-irradiated,liquid feed, which was sterilized by filtration.

FIG. 6 shows that irradiation, at approx. 30 kGy, does not negativelyimpact (a) cell growth (top panel); or, (b) protein production (bottompanel), while culturing cells in an irradiated test feed. Additionalsupplements 68 and 86 were also tested and showed similar results (datanot shown).

FIG. 7 shows that there is negligible impact of gamma irradiation onmicrosuspensions, and on dried, encapsulated microsuspensions. Thisgraph shows how encapsulated compounds (see far right) are impacted by30 kGy of gamma being either in microcapsules (“bead”) or asmicrosuspensions (MS) compared to non-irradiated liquid control. NG=nogamma irradiation, and G=gamma-irradiated.

FIG. 8 shows a schematic representation of a microencapsulated beadcomprising a labile component, which is embedded and/or engulfed inanti-oxidants to protect the labile molecule, by reducing the impact ofoxidation species generated during irradiation.

FIG. 9 shows chelation of reactive species and formation of exemplarycoordinate complexes of metal ions in trace elements with amino acids.

FIG. 10 shows a schematic representation of methods of preparingmicrosuspensions and encapsulated microsuspensions.

FIG. 11 shows two options for the direct addition of irradiated,microencapsulated beads to a bioreactor. Left panel shows beads addedwithin an autoclavable porous metal filter; or, right panel shows thatbeads can be added directly into the culture.

FIG. 12: Comparison of extended release of supplement introduced intoculture using the porous metal filter device for (a) microsuspensionbeads with PLGA coating: top panel, and (b) no beads, just AGT feed.

SUMMARY OF THE DISCLOSURE

The media, feed and supplement compositions described in this disclosurehave several desirable properties, which include but are not limited to,(i) ability to deliver certain components at “superconcentrated” levelsextending far beyond their normal solubility limits in a culture system,(ii) increased ability to maintain media/feed functionality even afterradiation sterilization, (iii) increased ability for extended release ofinternal components, (iv) high and quick solubility, (v) longer shelflife in dry format, (vi) increased thermostability, (vii) reduced riskof viral contamination up to 8 logs, (viii) the ability to be combinedwith other sterilizing technologies such as UV, filtration, and/or HTSTpasteurization, (ix) the ability to be applied to a variety of mediaformats such as AGT, DPM, APM, as well as, to formulations having labilecomponents at higher concentrations, (x) the ability to be applied to avariety of product types such as media, feeds, supplements, functionaladditives, etc. Due to these characteristics, the compositions can beadded directly into a bioreactor or into a culture already in progress,and thereby can improve customer workflows and bioreactor productivity.

Accordingly, the compositions, methods and uses described in thisdisclosure are directed, in part, to cell culture media, concentratedfeeds, functional additives, supplements that comprise amicrosuspension; may also be directed to a novel cell culture media,feed and/or supplement composition comprising one or more encapsulatedmicro and/or nanosuspensions; and may further be directed to sterilizingthe above compositions using radiation such that the functionality ofthe media/feed is maintained even after exposure to radiation. Thepresent disclosure also provides methods of making such media, includingcharacteristics of such media and its uses. Throughout this disclosure,some references may be made to cell culture media alone, but it wouldalso include feeds and/or supplements, as applicable.

In one embodiment, the disclosure is directed to a method of making amedia, feed or supplement composition, the method comprising: adding aminimal volume of an aqueous solution to a dry powder of the media, feedor supplement to make a paste; and mixing the paste vigorously toprepare a microsuspension.

In another embodiment, the disclosure is directed to a method ofpreparing a composition comprising a labile component comprisingoptionally, mixing in an effective amount of an anti-oxidant with themicrosuspension of the labile component to form a mixture; encapsulatingthe microsuspension or the mixture of step 2 in a suitable capsularmaterial such that a microcapsule or bead is formed; and drying themicrocapsule or bead. In one embodiment, the labile substance isattached to a dendrimer. In any of these embodiments, the capsularmaterial may be selected from the group comprising, for example,alginate, poly-L-lactic acid, chitosan, agarose, gelatin, hyaluronicacid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparinsulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, watersoluble cellulose derivatives and carrageenan.

In another embodiment of the method, the bead is further coated with amaterial that extends the release of the labile component from the bead.In a further aspect, the coating is selected from the group comprisingof, for example, poly-glycolic acid, PLGA (poly-lactic-co-glycolicacid), collagen, polyhydroxy-alkanoates (PHA), poly-ε-caprolactone,poly-ortho esters, poly-anhydrides, poly-phosphazenes, poly-amino acids,polydimethylsiloxane, polyurethranes, poly-tetrafluoroethylene,polyethylene, polysulphone, poly-methyl methacrylate,poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinylchloride, polystyrene and poly-vinyl pyrrolidone.

In one embodiment, the bead is irradiated, and in a further embodiment,the beads are irradiated with gamma-irradiation. In certain embodiments,the bead can be additionally irradiated with UV rays, whereupon, thebead may be free of PPV and MMV viruses.

In several embodiments, the media is a dry-format media, and the labilecomponent to be protected is selected from the group consisting of apolyamine, a growth factor, a cytokine and a vitamin.

In some embodiments, the capsular material is soluble uponreconstitution with an aqueous solvent. In other embodiments, theencapsulating matrix encapsulates a dendrimer-labile component complex.In some embodiments, the dendrimer is a polyamidoamine dendrimer, apolypropylenimine dendrimer, or a polypropylamine (POPAM) dendrimer.

In any of the above embodiments, the methods described can, for example,achieve one or more of the following with respect to the media, feeds,supplements, or functional additives: 1) make them more resistant toirradiation (as measured, for example, based on retetention of efficacyor functionality); 2) increase stability at ambient temperature; 3)allow for extended release of some components; 4) increase stability fortransport at ambient temperatures; 5) increase stability to temperaturefluctuations.

The compositions used in the methods above may comprise a powdered cellculture medium further comprising a labile compound, and/or at least oneconcentrated component.

The disclosure is also directed to the following compositions: media,feeds or supplement compositions comprising a microsuspension of amedia/feed component. In one embodiment, the medium, feed or supplementcomposition comprises a mixture of a labile component and ananti-oxidant that is microencapsulated within a capsular matrix into abead. In a further embodiment, the capsular material is selected fromthe group comprising, for example, alginate, poly-L-lactic acid,chitosan, agarose, gelatin, hyaluronic acid, chondroitin sulfate,dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate,gellan gum, xanthan gum, guar gum, water soluble cellulose derivativesand carrageenan. In another embodiment, the bead is coated with acoating solution. In yet another embodiment, the coating solution isselected form the group consisting of poly-glycolic acid, PLGA(poly-lactic-co-glycolic acid), collagen, polyhydroxyalkanoates (PHA),poly-ε-caprolactone, poly-ortho esters, poly-anhydrides,poly-phosphazenes, poly-amino acids, polydimethylsiloxane,polyurethranes, poly-tetrafluoroethylene, polyethylene, polysulphone,poly-methyl methacrylate, poly-2-hydroxyethylmethacrylate, polyamides,polypropylene, poly-vinyl chloride, polystyrene, poly-vinyl pyrrolidone,poly-L-lysine and polyornithine.

In a further embodiment, the composition further comprises a chelatedreactive species. In a further aspect of this embodiment, the reactivespecies are either cations, metals ions or trace elements. In anotheraspect, the chelating moieties are selected from the group comprisingEDTA, citrate, succinate, cyclodextrin, clatharates, dendrimers andamino acids.

In any of the compositions described above, the composition can beirradiated. In a further aspect, the irradiation may be with gamma-rays;and in a specific aspect, the gamma-rays may be about 25-100 kGy. In apreferred aspect, gamma-rays of 30-50 kGy are used. In a specificembodiment, the gamma-ray is 30 kGy. One of skill in the art will becapable of determining a level of irradiation that maximizes theobjective (for example reduction of live virus or other sterilization)while minimizing the impact on, for example, the functionality of themedia or the functionality of a particular component of the media (forexample, as measured by a component that is particularly sensitive toirradiation or whatever sterilization technique is being employed).

In one aspect of the invention, the methods described herein may becombined with additional step(s) the goal of which is to measure afunctional parameter of the media or composition after sterilization (orafter any final processing or finishing step). For example, afterirradiation, the sample may be subject to a functional assay formeasuring the impact of the irradiation on a particular component orgroup of components. This step may be used to confirm, for example, thatthe composition is suitably free of virus and that the components of themedia remain in satisfactory condition for the intended application.

In one embodiment, any of the medium, feed or supplement microsuspensionand/or encapsulated microsuspension compositions described above areadded aseptically, directly into a bioreactor. In another embodiment,any of the any of the medium, feed or supplement microsuspension and/orencapsulated microsuspension compositions described above are place in aporous metal cylinder, that is autoclavable, within a bioreactor.

Any of the media, feeds or supplements described above can beserum-free, protein free, or serum and protein-free. Any of the media,feeds or supplements described above may be specifically designed forsuspension cell culture, for mammalian cell culture, for insect cellculture, for hybridoma cell culture, for stem cell culture, for inducedpluripotent cell culture, for pluripotent cell culture, for tissueexplants and/or organ culture, for three-dimensional culture of cells onartificial cell matrices, and other cell and tissue culture applicationsthat one of skill in the art could adapt the teachings in thisdisclosure to.

Any of the media, feeds or supplements described above can comprise apowdered cell culture medium, and in a preferred embodiment, thepowdered cell culture medium is AGT (advanced granulation technologycell culture medium).

Any of the media, feeds or supplements described above can be used toproduce a dry format cell culture medium.

Any of the media, feeds or supplements described above can be increasethe shelf life of the cell culture medium.

Any of the media, feeds or supplements described above can be used forrecombinant protein production, for vaccine production, for cellproduction including stem cells, for biofuel production, or forproduction of nutrients.

Any of the media, feeds or supplements described above can be stored andhandled at ambient temperatures, can withstand temperature fluctuations,can withstand HTST and/or UHT pasteurization, can withstand exposure tosterilizing radiation wavelengths such as gamma, UV, and others know inthe art.

DETAILED DESCRIPTION

Reference will now be made in detail to various exemplary embodiments.It is to be understood that the following detailed description isprovided to give the reader a fuller understanding of certainembodiments, features, and details of aspects of the disclosure, andshould not be interpreted as a limitation of the scope of thedisclosure.

Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “advanced granulation technology” or AGT, as used in thisapplication refers to a process of preparing cell culture medium thatinvolves spraying one or more aqueous solutions onto air suspendedpowdered medium components, with gentle, rapid evaporation of water,under conditions where sensitive components do not lose their efficacy,resulting in an agglomerated granule and a homogenous distribution ofthe sprayed ingredients throughout the agglomerated granules. Thegranulated powder (AGT) is discussed in Fike et al., Cytotechnology,2006, 36:33-39, and in Applicants' patents and/or patent applications:U.S. Pat. No. 6,383,810, issued May 7, 2002; U.S. Pat. No. 7,572,632,issued Aug. 11, 2009; and in U.S. patent application Ser. No. 11/669,827filed Jan. 31, 2007, whose disclosures are hereby incorporated byreference in their entirety. Briefly, AGT media is a dry, powderedmedium that is highly desired in the industry, for properties like largeparticle size, reduced amount of fine dust while handling, highwetability, low dissolution times into solvent, auto-pH and autoosmolarity maintenance, etc.

Another type of dry powder format is the APM powder which is “advancedpowder media”, which has advantageous properties of a particle size thatis not as fine as DPM powder, but is not as large as AGT granules.

The term “susceptible compound” or “sensitive compound” or “labilecompound” as used in this application refers to substance, chemical orcompound to be protected from degradation or reaction with “reactivespecies” present in dry format media. Examples of such compounds in cellculture media include but are not limited to: ethanolamine, vitamins,cytokines, growth factors, hormones, etc.

The term “encapsulating agent” may sometimes be referred to as“sequestering agent” in this application, and refers to theencapsulation, protection, separation, or sequestering of susceptiblechemicals or components in the cell culture medium or feed, away fromconditions that enhance degradation, or reactivity with other reactivechemicals such as amino acids, trace metal elements such as manganese,copper, etc., inorganic buffers such as sodium bicarbonate and othersodium phosphates; and organic buffers such as MOPS, HEPES, PIPES, etc.,which may react slowly with the susceptible compound, thereby making thelabile component lose its desirable properties over time. Alternately,encapsulation, protection, separation, or sequestering may be done toprotect the susceptible chemical or component from physical damage suchas, radiation damage, or heat damage, or physical stress, from exposureto moisture/condensation, or from dehydration, etc. The terms “protect”or “separate” or “sequester” or “encapsulate” may have been usedinterchangeably in the disclosure, and convey the concept of protectingthe susceptible chemical or compound from degrading conditions orchemicals. The “soluble sequestering agent” itself may be soluble uponreconstitution with an aqueous medium, whereupon it releases the“sensitive” encapsulated material. Or, the “insoluble sequesteringagent” may be insoluble upon reconstitution with an aqueous medium,whereupon after releasing the “sensitive” encapsulated material, it canbe removed by means such as filtration, decanting, etc. from thereconstituted endproduct.

Examples of matrices that may be used for microencapsulation include butare not limited to, alginate, poly-L-lactic acid (PLL), chitosan,agarose, gelatin, hyaluronic acid, chondroitin sulfate, dextran, dextransulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthangum, guar gum, water soluble cellulose derivatives, carrageenan and soon.

Optionally, the microcapsules may be coated for one of several reasons:to extend and slowly release the microcapsule components; for protectionof labile components against any type of damage, say, radiation, heat,dehydration, etc. Coatings may include but are not limited to,poly-glycolic acid, PLGA (poly-lactic-co-glycolic acid), collagen,polyhydroxy-alkanoates (PHA), poly-ε-caprolactone, poly-ortho esters,poly-anhydrides, poly-phosphazenes, poly-amino acids,polydimethylsiloxane, polyurethranes, poly-tetrafluoroethylene,polyethylene, polysulphone, poly-methyl methacrylate,poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinylchloride, polystyrene, poly-vinyl pyrrolidone, etc.

Labile media or feed components include, but are not limited to,compounds such as vitamins, for example, thiamine, B12; amino acids likeglutamine; polyamines like ethanolamine; cytokines; growth factors, etc.

Agents used to chelate, deactivate or shut reactive molecules withinmedia include, but are not limited to, compounds such as EDTA, citrate,succinate, cyclodextrin, clatharates, dendrimers, amino acids, etc.

The cell culture medium is preferably a powdered cell culture medium. Inone embodiment, the powdered cell culture medium is an advancedgranulation technology (AGT) cell culture medium. The cell culture mediaalso refers to feeds, concentrated supplements, concentrated media, andin some instances, liquid media, as applicable.

The terms “cell culture” or “culture” as used in this application referto the maintenance of cells in an artificial (e.g., an in vitro)environment. It is to be understood, however, that the term “cellculture” is a generic term and may be used to encompass the cultivationnot only of individual prokaryotic (e.g., bacterial) or eukaryotic(e.g., animal, plant and fungal) cells, but also of tissues, organs,organ systems or whole organisms, for which the terms “tissue culture,”“organ culture,” “organ system culture” or “organotypic culture” mayoccasionally be used interchangeably with the term “cell culture.”

The term “cultivation” as used in this application refers to themaintenance of cells in an artificial environment under conditionsfavoring growth, differentiation, or continued viability, in an activeor quiescent state, of the cells. Thus, “cultivation” may be usedinterchangeably with “cell culture” or any of its synonyms describedabove.

The terms, “cell culture medium,” “culture medium,” or “medium” (and ineach case plural media) as used in this application refer to a nutritivecomposition that supports the cultivation and/or growth of cells. Thecell culture medium may be a complete formulation, i.e., a cell culturemedium that requires no supplementation to culture cells, may be anincomplete formulation, i.e., a cell culture medium that requiressupplementation or may be a medium that may supplement an incompleteformulation or in the case of a complete formulation, may improveculture or culture results. The terms “cell culture medium,” “culturemedium,” or “medium” (and in each case plural media) refer tounconditioned cell culture media that has not been incubated with cells,unless indicated otherwise from the context. As such, the terms “cellculture medium,” “culture medium,” or “medium” (and in each case pluralmedia) are distinguished from “spent” or “conditioned” medium, which maycontain many of the original components of the medium, as well as avariety of cellular metabolites and secreted proteins.

The term “powder” or “powdered” as used in this application refers to acomposition that is present in granular form, which may or may not becomplexed or agglomerated with a solvent such as water or serum. Theterm “dry powder” may be used interchangeably with the term “powder;”however, “dry powder” as used herein simply refers to the grossappearance of the granulated material and is not intended to mean thatthe material is completely free of complexed or agglomerated solventunless otherwise indicated.

A “1× formulation” refers to any aqueous solution that contains some orall ingredients found in a cell culture medium at workingconcentrations. The “1× formulation” can refer to, for example, the cellculture medium or to any subgroup of ingredients for that medium. Theconcentration of an ingredient in a 1× solution is about the same as theconcentration of that ingredient found in a cell culture formulationused for maintaining or cultivating cells in vitro. A cell culturemedium used for the in vitro cultivation of cells is a 1× formulation bydefinition. When a number of ingredients are present, each ingredient ina 1× formulation has a concentration about equal to the concentration ofthose ingredients in a cell culture medium. A “1× formulation” of theseamino acids contains about the same concentrations of these ingredientsin solution. Thus, when referring to a “1× formulation,” it is intendedthat each ingredient in solution has the same or about the sameconcentration as that found in the cell culture medium being described.The concentrations of ingredients in a 1× formulation of cell culturemedium are well known to those of ordinary skill in the art. See CellCulture Technology for Pharmaceutical and Cell-Based Therapies, 42-50(Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006),which is incorporated by reference herein in its entirety. Theosmolarity and/or pH, however, may differ in a 1× formulation comparedto the culture medium, particularly when fewer ingredients are containedin the 1× formulation.

This disclosure refers to microsuspensions and dried microcapsule beadswhere the concentration of the same ingredient is concentrated in themicro/nanosuspension, and is concentrated even further in a dryencapsulated bead format. Accordingly, a “7× formulation” is meant torefer to a concentration wherein each ingredient in thatmicro/nanosuspension or encapsulated bead is about 7 times moreconcentrated than the same ingredient in the corresponding liquid cellculture medium/feed or supplement. A “10× formulation” is meant to referto a concentration wherein each ingredient in that micro/nanosuspensionor encapsulated bead is about 10 times more concentrated than the sameingredient in the liquid cell culture medium/feed or supplement. As willbe readily apparent, “5× formulation,” “25× formulation,” “50×formulation,” “100× formulation,” “500× formulation,” and “1000×formulation” designate formulations that contain ingredients at about 5to 25-, 25-50-, 50-70-, 70-100-, 100-500-, 500-1000-fold concentrations,respectively, as compared to a 1× cell liquid medium, feed orsupplement. Again, the osmolarity and pH of the media formulation andconcentrated solution may vary. A formulation may contain components oringredients at 1× with respect to a particular cell culture protocol,but at a concentration, for example, 2, 2.5, 5, 6.7, 9, 12 etc. X withrespect to a different culture protocol or different base medium.

Microsuspensions

Nutrient feeds, functional additives or supplements are generallyprovided as clear liquid concentrates or as powders that getreconstituted into clear liquid concentrates for delivery directly intothe bioreactor. This means that the components therein are never beyondtheir solubility limits. If they are prepared beyond their solubilitylimits, it is well known that precipitate forms, either as flakes orfine precipitates, usually white cloudiness in the bottle. Settling ofthese components occur in several hours, which means that theconcentrated solution cannot be used to deliver accurate amounts offeed.

This disclosure provides techniques for preparing a medium, feed and/orsupplement components in such a way that the concentrated components donot precipitate out. This is achieved by making a microsuspension and/ornanosuspension (also referred to as micro/nanosuspension) from a drypowder cell culture medium or feed that has one or more concentratedcomponent.

A micro/nanosuspension is a micron/nano-sized solid in an aqueoussolvent base that, in one embodiment, does not separate over time.Micro/nanosuspensions, for example, provide a means of concentrating oneor more media/feed components beyond the solubility limit of thatcomponent. Some desirable properties of micro/nanosuspensions include,but are not limited to, enabling increased nutrient supplementconcentrations (e.g., amino acids) in minimal volume; extremely rapiddissolution of micro/nanosuspensions components in aqueous solutions(more rapid than the media would dissolve absent such preparation);capacity for encapsulation (i.e. for sterilization and protection ofcomponents in encapsulated form); capacity for direct addition ofsterile, micro/nanosuspensions beads into pre-existing cultures in abioreactor; the ability to increase efficiency and manufacturingprocesses in a bioreactor.

Microsuspensions have been prepared in other industries, for example, inthe pharmaceutical or cosmetic industry, generally using two approaches:i) a top-down approach, and ii) a bottom-up approach. In the top-downapproach, the particles of a dry powder are broken down by a millingprocess such as a Fitz® mill (the industry standard for precise particlesize reduction), until microsuspensions are obtained, or by wet-millingor by using a microfluidizer to obtain nanosuspensions. In the bottom-upapproach, components within a solution are gradually precipitated out bygentle manipulation of parameters like pH, or polymerization parameters,until micro/or nanosuspensions are obtained. These methods were notuseful in preparing the micro/or nanosuspensions described here. Giventhe sensitive nature of the media and supplements utilized herein,applicants recognized the need for a novel method for preparing thesemicro/or suspensions of media, feeds. As described in Example 1 and inFIG. 1, applicants started with a dry media, feed or supplement powder,and by adding a minimal amount of WFI water to the dry powder,vigorously mixing the slurry into a homogenous paste such that allparticles were coated with an aqueous phase, micro/nanosuspensions wereformed. As one of skill in the art would know in light of the disclosureherein, any aqueous base beside water, for example, buffer, balancedsalt solution, a liquid medium, or any solution comprising one or moremedia components including amino acids, lipids, etc., may be added toany powdered formulation to make a micro/nanosuspension described inthis disclosure. One of skill may further adapt any of the steps ormaterials used in the suggested protocol: for instance, the sequence ofaddition steps, the sequence and number of mixing steps, the volume ofliquid, the mixing time, the apparatus or device for mixing the slurryto homogeneity, the media or feed formulation, etc. and they would knowhow to manipulate the conditions to suit the consistency and nature ofthe desired micro/nanosuspension.

In one embodiment, any structural or informational molecule, anynutrient, required by a cell in culture can be made into amicro/nanosuspension using the techniques, or variations of thetechniques described here. Besides media and feeds, examples ofstructural and/or informational molecules may include, but are notlimited to, vitamins, amino acids, peptides, macromolecules,anti-oxidants, hormones, growth factors, and so on. In certainembodiments, micro/nanosuspensions of cellular scaffolding matricescomplexed with cellular nutrients may be made in order to grow cellswithin the suspensions three dimensionally.

The micro/nanosuspension compositions prepared as described above may beuseful in many applications, for example, in nutrient supplementation tosignificantly boost component concentrations beyond the level ofsolubility of the component in question, such that, volume of additionto the reactor is minimal; or, for encapsulating the microsuspensions,as described below, and making a dried form of the encapsulated beadresulting in a “super concentrated” supplement that can be directlyadded to the bioreactor, which has not been done before.

In one embodiment, microsuspensions can be made from any form of drypowder, of any component that needs to be provided in culture in aconcentrated form, and provide at least a 2 to 5-fold, 5 to 10-fold, 10to 15-fold, 15 to 20-fold, 20 to 25-fold, 25 to 30-fold, 30 to 50-fold,50 to 70-fold, 70 to 100-fold concentration of the component in themicrosuspension over an equivalent liquid concentrate or feed having thesame component in solution.

Microencapsulation

This disclosure provides a method of microencapsulating themicro/nanosuspension described above, that was made from a dry powderedcell culture medium, feed, supplement or concentrate. The resultingencapsulated products may be referred to as ‘microcapsules’,‘encapsulated bead’, ‘beads’, ‘capsules’ or ‘microbeads’ in thisdisclosure. When the encapsulated micro/nanosuspension is dried intobeads, the drying step provides a greater degree of concentration of theencapsulated micro/nanosuspensions. Microencapsulation may be done, forexample, to “keep apart” or sequester sensitive or labile components ina complex mixture such as cell culture media/feed. Thus, encapsulationmay yield higher concentrations of certain feed components such as, forexample, amino acids, so that these feeds can be directly added asconcentrated, high nutrient supplements into any culture system, forexample, in fed-batch cultures. Further coating of the capsule mayaffect delayed-release of nutrients to into cell cultures (discussedbelow). Encapsulation can be done by: (a) a standard microencapsulationprocess of microsuspensions and nanosuspensions for “gently-releasing”some or all components over several hours; (b) an alternativebead-gelling process to significantly retard the internal componentrelease. An exemplary demonstration of delayed release can be found inthe Examples and in FIGS. 3, 4 and 8.

In one specific embodiment, the agent used to encapsulate or embed thelabile component was alginate. Alginate microcapsules have been used formany purposes, including drug delivery and the immobilization of cellsgrowing in cell culture to enhance cell growth and viability. See e.g.,Serp et al., Biotechnology and Bioengineering, 2000, 70(1):41-53;Breguet et. al., Cytotechnology, 2007, 53:81-93; Chayosumrit et al.,Biomaterials, 2010, 31:505-14; U.S. Pat. No. 7,482,152; and U.S. Pat.No. 7,740,861, all of which are incorporated by reference in theirentirety.

The encapsulation technique was also described in Applicants' co-pendingapplication, PCT/US2012/024194, which described entrapping certainlabile, sensitive or susceptible compounds such as ethanolamine,vitamins, growth factors like insulin, etc. in capsular materials,including but not limited to, alginate.

Without intending to be bound by any theory, it appears thatencapsulating or embedding sensitive components within another moleculereduces the labile compound's direct contact with other components orconditions that promote its degradation, or reduces its stability.Methods describing the preparation of microcapsules for the reduction ofethanolamine degradation by microencapsulation is described inApplicant's co-pending application, PCT/US2012/024194, filed Feb. 7,2012, whose disclosure is incorporated by reference herein in itsentirety. Although those methods were primarily exemplified within thecontext of ethanolamine stabilization, they can be used/adapted tostabilize any susceptible or labile chemical or compound in a media,feed or supplement. It is understood that the microencapsulation methodsdescribed therein can be used for stabilizing any susceptible compoundrequired for cell culture, including but not limited to, vitamins likethiamine, B12, etc., unstable amino acids such as glutamine, cytokines,growth factors, sensitive and valuable proteins or peptides, etc. andfor enhanced delivery of the stabilized compound, and can be applied tofields beyond cell culture media development. In this disclosure, theencapsulation technique was adapted to micro/nanosuspension beads, whichrequired adaptation of several steps and techniques. For instance, theentrapping steps for susceptible compounds in the PCT/US2012/024194application lacked several steps. For encapsulation of microsuspensions,the capsular material, such as alginate, was mixed and blended with themicrosuspension. This mix was then aspirated into a dispensing devicesuch as a pipette or a dropper and droplets of encapsulatedmicrosuspension were gradually generated by dropping the mix gently ontoa non-stick surface, for example, on parafilm. Then, a cross-linkingagent was added to the drop to form beads. These beads were desiccatedand vacuum dried to remove moisture, and are generally referred to as“encapsulated microsuspension beads” or just “beads”.

As one of skill in the art would know based on the instant disclosure,one may adapt any of the steps or the materials used in the suggestedprotocol (see Example 2). For instance, a variety of capsular materialsmay be used, or a variety of drop delivery devices including pipettes,droppers, syringes or any adaptation thereof may be used, or anycross-linking agent may be used, or the bead may be dried or desiccatedby a variety of means and to differing degrees of dryness and/orhardness. One of skill in the art will be able to determine appropriateencapsulating agents for the purpose at hand, for instance, alginate,poly-L-lactic acid (PLL), chitosan, agarose, gelatin, hyaluronic acid,chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate,heparan sulfate, gellan gum, xanthan gum, guar gum, water solublecellulose derivatives, carrageenan and so on.

Microcapsules are typically spherical particles having a diameter of 2mm or less, usually within the diameter range of 0.05-1.5 mm. Typically,alginate microcapsules are formed by crosslinking between thepolyanionic alginate and a divalent or trivalent polyvalent cation, suchas calcium chloride. Other salts for cross-linking may be divalent ortrivalent cations, such as magnesium chloride, barium chloride, andaluminum sulfate.

Encapsulation has several advantages, some of which include, but are notlimited to, protection of labile components from degradation, or fromunwanted reactions; or to delay and/or extend the release-time of theencapsulated components into cell culture In one embodiment, protectiondue to microencapsulation of media; or to increase the stability andstorage of cell culture media, feeds and supplements comprising labilecompounds at ambient temperatures. The encapsulated compound can bedried into beads, which can then be blended and/or mixed with othermedia components. Accordingly, micro/nanosuspensions may result in a1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-50%,50-60%, 60-70%, 70-80%, 80-90%, or 90-100% reduction in any loss ofmedia/labile component functionality, which may be measured by asuitable functional assay for the encapsulated labile or mediacomponent, using techniques known in the art, including the methodsdisclosed in this application. Examples of functional assays may be, theability of a media/feed composition comprising microcapsules to increasethe cell viability over days, or the cell number in a culture system, orrecombinant protein production, or an increase in the amount and/or thefunction of a recombinant protein being expressed (for example, anenzyme or a receptor functional assay, or the stability of anencapsulated labile component like glutamine can be evaluated duringculture, etc., as would be known to one of skill in the art).

In one embodiment, a sequestering agent like alginate was used toencapsulate or embed an ethanolamine-dendrimer complex. Dendrimers arehyper-branched synthetic macromolecules that can be made usingcontrolled sequential processes to give them defined structural andmolecular weight characteristics; reviewed in Astruc et al., Chem. Rev.2010, 110:1857-1959, which is hereby incorporated by reference in itsentirety. Dendrimers can be used to prepare the encapsulatedmicrosuspensions of the instant invention as well. In anotherembodiment, the dendrimer used in the methods as described inPCT/US2012/024194 was poylamidoamine, and it may be adapted for used inencapsulated micro/nanosuspensions. Other dendrimers that can be used inthe methods described in this application include, but are not limitedto polypropylenimine (PPI) dendrimers, phosphorous dendrimers,polylysine dendrimers, polypropylamine (POPAM) dendrimers,polyethylenimine dendrimers, iptycene dendrimers, aliphatic poly(ether)dendrimers, or aromatic polyether dendrimers.

In one embodiment, microencapsulated micro/nanosuspensions can be madefor any component that needs to be provided in culture in a concentratedform, and provides at least a 2 to 5-fold, 5 to 10-fold, 10 to 15-fold,15 to 20-fold, 20 to 25-fold, 25 to 30-fold, 30 to 50-fold, 50 to70-fold, 70 to 100-fold concentration of the component in theencapsulated micro/nanosuspension over an equivalent liquid concentrateor feed having the same component in solution. In one exemplaryembodiment, the microsuspension preparation of a concentrated feedpreparation as seen in the Figures was about 7-fold more concentratedthan its corresponding liquid feed, whereas the dried encapsulated formof the same microsuspension was about 10-fold more concentrated than itscorresponding liquid feed.

Anti-Oxidants

This disclosure also describes that the labile compounds within thecapsule may be further combined with one or more protective substances,such as anti-oxidants, prior to the encapsulation process. Therefore, incertain embodiments, a mixture anti-oxidant and a labile component in apowdered media, feed, or supplement may be used as a starting materialto prepare the microsuspension preparation, prior to encapsulation.Exemplary anti-oxidants include, but are not limited to, vitamins likeascorbic acid, beta-carotene, vitamin A, E, lycopene, flavanoids,selenium, etc. Effects of anti-oxidants on protection due to irradiationis depicted in FIG. 8.

In one embodiment, protection due to the anti-oxidants prior tomicroencapsulation may result in a 1-5%, 5-10%, 10-15%, 15-20%, 20-25%,25-30%, 30-35%, 35-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or90-100% reduction in loss of media/labile component functionality, whichmay be measured by a suitable functional assay for the labile or mediacomponent. The dried microcapsule beads can then be blended into and/ormixed with other media components.

Chelation

This disclosure also describes chelation agents, which are agents thatchelate, deactivate or shut off reactive molecules found within mediathat include cations, metals ions, trace elements, etc. Reactivemolecules interact adversely with labile compounds in media and reducetheir efficacy. By chelating/complexing the reactive compounds,compounds such as EDTA, citrate, succinate, cyclodextrin, clatharates,dendrimers, amino acids, etc. reduce their reactivity. In addition,chelates may be designed to remain partitioned outside amicrocapsule/bead comprising a labile compound. The effect of chelationon the protection of labile compounds is described in FIG. 9.

In one embodiment, protection due to chelation of reactive and/or ROSproducing media/feed components outside the microcapsule may result in a1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-50%,50-60%, 60-70%, 70-80%, 80-90%, or 90-100% reduction in loss ofmedia/labile component functionality, which may be measured by asuitable functional assay for a labile or media component. Examples offunctional assays may be, the ability of a media/feed compositioncomprising microcapsules and chelated components to increase the cellviability over days, or the cell number in a culture system, orrecombinant protein production, or an increase in the amount and/or thefunction of a recombinant protein being expressed (for example, anenzyme or a receptor functional assay, or the stability of anencapsulated labile component like glutamine can be evaluated duringculture, etc., as would be known to one of skill in the art).

Delayed-Release

In one embodiment, optionally, the microcapsule can further be coatedwith protective coatings like PLGA for delayed-release orextended-release of components within the microcapsule. Typical mediumcomponents for delayed release may include, but are not limited to,vitamins, glucose, amino acids, growth factors or cytokines. Componentscan be released from within the same microcapsule at the same rate, orreleased from different microcapsules at different rates and atdifferent times, depending on the delayed-release formulation. Anexemplary use for such a dual-format, delayed release medium would be inthe release of growth components (for e.g., glucose, amino acids, etc.)early in the culture followed by release of productivity components (fore.g., recombinant protein expression inducers such as galactose, etc.)later into the post-log phase of the culture. Such a medium would needno customer action as the dual-format medium would take care of thetimely release of necessary components. Another example would be inmaintaining two or more components separate until a designated time whenthey need to react together within a culture for a specific purpose.

Coating solutions that may be used to coat after microencapsulation, maybe applied to provide additional layers, and include, but are notlimited to, poly-glycolic acid, PLGA (poly-lactic-co-glycolic acid),collagen, polyhydroxyalkanoates (PHA), poly-ε-caprolactone, poly-orthoesters, poly-anhydrides, poly-phosphazenes, poly-amino acids,polydimethylsiloxane, polyurethranes, poly-tetrafluoroethylene,polyethylene, polysulphone, poly-methyl methacrylate,poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinylchloride, polystyrene, poly-vinyl pyrrolidone, poly-L-lysine orpolyornithine. Encapsulated components that are formed into beads andexternally coated may themselves be further encapsulated so that a widerange of release options are available depending upon the coatingcharacteristics of the surrounding coating and the individual beadcoatings.

The additional outer coating may be selected from alginate,poly-L-lactic acid, chitosan, agarose, gelatin, hyaluronic acid,chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate,heparan sulfate, gellan gum, xanthan gum, guar gum, water solublecellulose derivatives and carrageenan. Effects of coating aredemonstrated and discussed in the Examples and in FIGS. 3, 4 and 8. Inone embodiment, delayed-release of internal components from amicrocapsule may result in maintenance of the encapsulated component ina cell culture system over 6-24 hours, 24-48 hours, 48-72 hours, over 1day, 2 days, 3-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days,25-30 days, 30-40 days, 40-50 days, over the duration of the culture.Delayed release may be measured by a suitable time-release assay for alabile or media component within the capsule, and may optionally becoated for extended release as well. An exemplary time release assay isdiscussed below. Other examples of functional assays may be, the abilityof a media/feed composition comprising coated microcapsules to increasethe cell viability over days, or the cell number in a culture system, orrecombinant protein production, or an increase in the amount and/or thefunction of a recombinant protein being expressed (for example, anenzyme or a receptor functional assay, or the stability of anencapsulated labile component like glutamine can be evaluated duringculture, etc., as would be known to one of skill in the art).

Besides the typical medium components like vitamins, glucose, aminoacids, growth factors or cytokines, the following cell culturenutrients/reagents may also be targeted for delayed-release. Theyinclude, but are not limited to, all chemicals or compositions that areused to promote cell metabolism whether for growth or product synthesisand secretion such as glucose and other forms of hexose, amino acids,salts, peptides, proteins such as collagen and protein fractions,lipids, hormones, vitamins, nucleotides/nucleosides, trace elements,ribonucleotides/ribonucleosides, sera, sera fractions such as bovinealbumin and ceruloplasmin, and other components that are not directlymetabolized but provide cell culture-related advantages to cells inculture, such as pluronic of various types such as F68 which preventcell clumping, fibronectin and peptide sequences such as RGD to supportcell attachment and anti-foam reagents. In addition to single ormultiple individual components, mixtures of components may be includedin this delay-release methodology. The powder itself may be either infine-milled or granulated (AGT) format.

An Exemplary Assay to Detect Delayed-Release

Detection of release-delay involves assaying over a period of time forthe component(s) in question and observing an increase over time. Anexample for a typical CHO cell nutrient supplement would be measuringfor glucose. The supplement containing glucose would be added to waterand a T=0 sample withdrawn and held. Then at subsequent time periods(such at 24, 48, 72 hours) additional samples can be withdrawn. If therewere a delayed-release component in the supplement, then glucose wouldbe seen rising over time. One assay for measuring glucose would bemeasurement of glucose oxidase, which is commercially available in kitformat. The principle here is that glucose oxidase reacts with glucoseto yield gluconic acid and hydrogen peroxide. The hydrogen peroxidereacts with o-dianisidine to yield oxidized o-dianisidine, which forms apink color upon exposure to sulfuric acid. This can be readspectrophotometrically. Another assay for glucose measurement or forother supplement components such as amino acids would be by HPLC. Themethod would measure a continual increase of the release component overtime, which is the hallmark of delayed-release.

Sterilization

In this disclosure, a labile, sensitive or susceptible compound includesbut is not limited to substances sensitive to physical, chemical,radiation degradation/destruction. Typical sensitive media substancesmay be sensitive to chemical interaction with a reactive oxygen species,sensitive to metals including trace metals, sensitive to hightemperature, to irradiation (gamma, X-ray, UV, ionizing, etc.), tofreezing temperatures, to freeze-thaw, to pressure, agitation, etc. In afurther aspect of this embodiment, trace elements or substancesgenerating reactive oxygen species (ROS), etc. may be chelated and/orkept on the exterior of the microcapsule comprising the labilecomponent.

This disclosure provides a means for sterilizing a media comprisingencapsulated components using irradiation while protecting theirradiation labile components, for example, through anti-oxidant coatingand/or subsequent encapsulation. Sterilizing irradiation includeswavelengths of light such as gamma rays, UV rays, and other ionizingradiation. Sterilizing gamma irradiation used may be from 5 kGy to 100kGy. In one embodiment, the medium comprising encapsulated componentscan be sterilized by gamma irradiation of up to about 50 kGy withoutloss of media or feed functionality, and/or with about 8-log reductionof virus. This satisfies a SAL (Sterility Assurance Level) of >10 e⁻⁸ ofthe porcine parvo virus (PPV) and/or the murine minute virus (MMV). Insome embodiments, the medium comprising encapsulated components can besterilized by a combination of gamma irradiation greater than 50 kGy upto 100 kGy without loss of media or feed functionality.

In certain embodiments, about 6-8 logs of SAL, or reduction of viruseslike MMV and PPV were observed. In combination with other sterilizationtechnologies such as UV irradiation, further reduction of SAL waspossible, up to about 8 logs of SAL without loss of media or feedfunctionality. In addition, the reconstituted media can be pasteurizedby techniques such as HTST (high temperature short time) or UHT (ultrahigh temperature), and/or by filtration through anti-viral filters ofpore size 0.1-0.2 μm, which may provide further reduction of virusessuch as vesivirus, porcine circovirus, and other small enveloped virusesthat are problematic in the biotechnology industry, without loss ofmedia or feed functionality.

Due to their ability to be sterilized, and because there is no loss ofmedia or feed functionality, the compositions of this disclosure wouldfit into an enhanced cell culture workflow and improve bioreactorproductivity as they can added into a bioreactor directly during thepre-existing culture of a cell. In certain embodiments, the addition canbe either as encapsulated beads, or in other embodiments, additions maybe as micro/nanosuspensions. Another example would be in the directaddition of the sterile microcapsules into the bioreactor, addition offiltered water, mixing and then addition of cells.

Microcapsules could be used for nutrient supplementation in bioreactorsin at least two ways: 1) Sterile microcapsules could be added directlyto the bioreactor where they release components in a time-release manner(right), or 2) they may be added to a porous metal cylinder, cage, orany similar apparatus, with about 0.22μ pore size, keeping the beadsaway from contacting the cells in culture. With option 1, the beads mayinterfere with filtration, or may reduce cell count or productivity.With option 2, bioreactor stirring alone is enough to dissolve nutrientsupplement from inside the porous metal cage and to pass into thebioreactor.

Cell Culture Media

A cell culture medium is composed of a number of ingredients and theseingredients vary from one culture medium to another. A cell culturemedium may be a complete formulation, i.e., a cell culture medium thatrequires no supplementation to culture cells, may be an incompleteformulation, i.e., a cell culture medium that requires supplementationor may be a supplement that is used to supplement an incompleteformulation or in the case of a complete formulation, may improveculture or culture results.

Generally, upon reconstitution, a cell culture medium will have solutesdissolved in solvent. The solutes provide an osmotic force to balancethe osmotic pressure across the cell membrane (or wall). Additionallythe solutes will provide nutrients for the cell. Some nutrients will bechemical fuel for cellular operations; some nutrients may be rawmaterials for the cell to use in anabolism; some nutrients may bemachinery, such as enzymes or carriers that facilitate cellularmetabolism; some nutrients may be binding agents that bind and bufferingredients for cell use or that bind or sequester deleterious cellproducts.

Depending on the cell and the intended use of the cell, the ingredientsof the cell culture medium will optimally be present at concentrationsbalanced to optimize cell culture performance. Performance will bemeasured in accordance with a one or more desired characteristics, forexample, cell number, cell mass, cell density, O2 consumption,consumption of a culture ingredient, such as glucose or a nucleotide,production of a biomolecule, secretion of a biomolecule, formation of awaste product or by product, e.g., a metabolite, activity on anindicator or signal molecule, etc. Each or a selection of theingredients will thus preferably optimized to a working concentrationfor the intended purpose.

A basal medium is typically used for maintenance of a cell culture, andcan comprise a number of ingredients, including amino acids, vitamins,organic and inorganic salts, sugars and other components, eachingredient being present in an amount which supports the cultivation ofthe cell in vitro.

The media described herein that comprises microsuspensions and/orencapsulated microsuspensions can be a 1× formulation or can beconcentrated, for example, as a 5×, 10×, 20×, 50×, 500×, or 1000× mediumformulation. If the individual medium ingredients are prepared asseparate concentrated solutions, an appropriate (sufficient) amount ofeach concentrate is combined with a diluent to produce a 1× mediumformulation. Typically, the diluent used is water but other solutionsincluding aqueous buffers, aqueous saline solution, or other aqueoussolutions may be used. The media could be a basal media to whichadditional components need to be added, or a complete media whichrequires no additional additives and is capable of growing cells oncereconstituted.

In one embodiment, the reconstituted media from the dry powder thatcomprises microsuspensions and/or encapsulated microsuspensions resultsin an auto-pH and/or auto-osmolality medium/feed in that, it hasbalanced buffer concentrations and/or salt concentrations thatcontribute to automatically achieving a desired pH and osmolalitysuitable for growing a certain cell type without additional pH or saltconcentration adjustment.

In another embodiment, or in a further embodiment, the reconstitutedmedia from the dry powder that comprises microsuspensions and/orencapsulated microsuspensions results in a chemically defined cellculture medium. The presence of media proteins makes purification ofrecombinant protein difficult, time-consuming, and expensive and canalso lead to reduced product yields and/or purity. Thus, in oneembodiment, the cell culture medium would be serum-free andprotein-free, yet complete, such that it can support the growth of aparticular cell type. Alternately, the reconstituted media from the drypowder could be serum-free but still contain proteins derived from oneor more non-animal derived sources (animal origin free-AOF) like fromplants, yeast, algal, fungal, or recombinant sources such as bacteria,fungal, plant, yeast algal, etc. in the form of hydrolysates, or in theform of a purified protein or an hydrolysate fraction. In otherinstances, serum-free media may still contain one or more of a varietyof animal-derived components, including albumin, fetuin, varioushormones and other proteins. In another embodiment, the medium or mediasupplement is protein free and further, does not contain lipids,hydrolysates, or growth factors.

The media or supplements of this disclosure compr used for fed-batchcultivation. Fed-batch cultivation of cells is typically used forindustrial production of biomolecules, such as proteins to increase cellconcentration and to extend culture lifetime for a high productconcentration and volumetric productivity. Fed-batch cultures involvethe controlled addition of one or more nutrients, such as glucose, to abasal medium. The nutrient(s) help to control the growth of the cellculture by preventing nutrient depletion or accumulation and byproductaccumulation, thereby maintaining important parameters, such asosmolality and CO2 concentration, within levels that promote cell growthor minimize cell death for optimal product expression.

Cells and Viruses

Media/feeds containing microsuspensions and/or encapsulatedmicrosuspensions, as described herein, can also be used to culture avariety of cells. In one embodiment, the media is used to cultureeukaryotic cells, including plant or animal cells, such as mammaliancells, fish cells, insect cells, algal cells, amphibian cells or aviancells or to produce viruses, virus-like particles.

Mammalian cells that can be cultured with the media/feeds describedherein include primary epithelial cells (e.g., keratinocytes, cervicalepithelial cells, bronchial epithelial cells, tracheal epithelial cells,kidney epithelial cells and retinal epithelial cells) and establishedcell lines and their strains (e.g., 293 embryonic kidney cells, BHKcells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells,Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI 2650cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-Icells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2cells, LLC-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells,and TH-I, B1 cells, or derivatives thereof), fibroblast cells from anytissue or organ (including but not limited to heart, liver, kidney,colon, intestines, esophagus, stomach, neural tissue (brain, spinalcord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, andfibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells,IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempseycells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38cells, WI-26 cells, MiC11 cells, CHO cells, CV-1 cells, COS-1 cells,COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells,F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C3H/IOTI/2 cells, HSDM1C3 cells, KLN2O5 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK-(Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells,Swiss/3T3 cells, Indian muntjac cells, SIRC cells, CII cells, and Jensencells, or derivatives thereof).

Eukaryotic cells, including algal cells, may also be cultivated in themedia compositions of this disclosure comprising microsuspensions and/orencapsulated microsuspensions, to produce biofuels, under suitableconditions for growth and biofuel production.

Cells supported by the culture medium described herein can also bederived from any animal, preferably a mammal, and most preferably amouse or a human. Cells cultured according to the methods disclosedherein may be normal cells, diseased cells, transformed cells, mutantcells, somatic cells, germ cells, stem cells, precursor cells orembryonic cells, any of which may be established or transformed celllines or obtained from natural sources. Cells may be used forexperimental purposes or for production of useful components.

In one embodiment, the media described herein is used to culture ChineseHamster Ovary (CHO) cells, including recombinant CHO cells orCHO-derived cell lines like CHOS, CHOK1, DG44, RevO, etc. The term CHOcell includes reference to recombinant CHO cells and to all CHO-derivedcell lines described. CHO cells have been classified as both epithelialand fibroblast cells derived from the Chinese hamster ovary. A cell linestarted from Chinese hamster ovary (CHO-K1) (Kao, F.-T. And Puck, T. T.,Proc. Natl. Acad. Sci. USA 60: 1275-1281 (1968) has been in culture formany years but its identity is still not confirmed. Mostbiopharmaceuticals currently produce proteins in CHO cells for manyadvantages that the cell line has, such as human-like glycosylationpatterns, precise post-translation modification and low risk fortransmission of human viruses.

Cultivation of Cells

Cells supported by the culture medium described herein can be cultivatedaccording to the experimental conditions determined by the investigator.It is to be understood that the optimal plating and culture conditionsfor a given animal cell type can be determined by one of ordinary skillin the art using only routine experimentation. For routine monolayerculture conditions, using the cell culture media described herein, cellscan be plated onto the surface of culture vessels without attachmentfactors. Alternatively, the vessels can be precoated with natural,recombinant or synthetic attachment factors or peptide fragments (e. g.,collagen, fibronectin, vitronectin, laminin and the like, or natural orsynthetic fragments thereof), which are available commercially forexample from Life Technologies, Corp. (Carlsbad, Calif. R&D Systems,Inc. (Rochester, Minn.), Genzyme (Cambridge, Mass.) and Sigma (St.Louis, Mo.). Cells can also be seeded into or onto a natural orsynthetic three-dimensional support matrix such as a preformed collagengel or a synthetic biopolymeric material. For suspension cultivation,cells are typically suspended in the culture media described herein andintroduced into a culture vessel that facilitates cultivation of thecells in suspension, such as a spinner flask, perfusion apparatus, orbioreactor. Ideally, agitation of the media and the suspended cells willbe minimized to avoid denaturation of media components and shearing ofthe cells during cultivation.

The cell seeding densities for each experimental condition can beoptimized for the specific culture conditions being used. For routinemonolayer culture in plastic culture vessels, an initial seeding densityof 1-5×10⁵ cells/cm2 is preferable, while for suspension cultivation ahigher seeding density (e. g., 5-20×105 cells/cm2) may be used.

Mammalian cells are typically cultivated in a cell incubator at about37° C. The incubator atmosphere should be humidified and should containabout 3-10% carbon dioxide in air, more preferably about 8-10% carbondioxide in air and most preferably about 8% carbon dioxide in air,although cultivation of certain cell lines may require as much as 20%carbon dioxide in air for optimal results. Culture medium pH shouldgenerally be in the range of about 6.2-7.8, preferably about 7.1-7.4,and most preferably about 7.1-7.3. The cells may be cultured underdifferent conditions (pH, temperature and/or carbon dioxide) to enhanceprotein production.

Cells in closed or batch culture should undergo complete medium exchange(i. e., replacing spent media with fresh media) when the cells reach adensity of about 1.5-2.0×106 cells/ml. Cells in perfusion culture (e.g., in bioreactors or fermenters) will receive fresh media on acontinuously recirculating basis.

Virus and Vaccine Production

In addition to cultivation of cells in suspension or in monolayercultures, the present media may be used in methods for producing virusesfrom mammalian cells. Such methods comprise (a) contacting a cell (e.g.,a mammalian cell) with a virus under conditions suitable to promote theinfection of the cell by the virus; and (b) cultivating the cell in theculture media described herein under conditions suitable to promote theproduction of virus by the cell. The cell may be contacted with thevirus either prior to, during or following cultivation of the cell inthe culture media. Optimal methods for infecting a mammalian cell with avirus are well-known in the art and will be familiar to one of ordinaryskill. Virus-infected mammalian cells cultivated in the culture mediadescribed herein may be expected to produce higher virus titers (e. g.,2-, 3-, 5-, 10-, 20-, 25-, 50-, 100-, 250-, 500-, or 1000-fold highertiters) than cells cultivated in a cell culture media other than thecell culture media described herein.

These methods may be used to produce a variety of mammalian viruses andviral vectors, including but not limited to adenoviruses,adeno-associated viruses, retroviruses and the like, and are mostpreferably used to produce adenoviruses or adeno-associated viruses.Following cultivation of the infected cells in the culture mediadescribed herein, the used culture media comprising viruses, viralvectors, viral particles or components thereof (proteins and/or nucleicacids (DNA and/or RNA)) may be used for a variety of purposes, includingvaccine production, production of viral vectors for use in celltransfection or gene therapy, infection of animals or cell cultures,study of viral proteins and/or nucleic acids and the like.Alternatively, viruses, viral vectors, viral particles or componentsthereof may optionally be isolated from the used culture mediumaccording to techniques for protein and/or nucleic acid isolation thatwill be familiar to one of ordinary skill in the art.

Recombinant Protein Production

The present culture media may also be used in methods for the productionof recombinant proteins from cells, including eukaryotic cells grown insuspension, but preferably mammalian cells, and particularly frommammalian cells grown in suspension. Methods of producing a polypeptideaccording to the disclosure comprise cultivating a cell (e.g., amammalian cell) that has been genetically engineered to produce apolypeptide in the culture media described herein under conditionssuitable for expression of the polypeptide by the cell. Optimal methodsfor genetically engineering a mammalian cell to express a polypeptide ofinterest are well-known in the art and will therefore be familiar to oneof ordinary skill Cells may be genetically engineered prior tocultivation in the media of the disclosure, or they may be transfectedwith one or more exogenous nucleic acid molecules after being placedinto culture in the media. Genetically engineered cells may becultivated in the present culture media either as monolayer cultures, ormore preferably as suspension cultures according to the methodsdescribed above. Following cultivation of the cells, the polypeptide ofinterest may optionally be purified from the cells and/or the usedculture medium according to techniques of protein isolation that will befamiliar to one of ordinary skill in the art.

EXAMPLES Example 1: Protocols for Making Microsuspensions and/orNanosuspensions

Provided herein is a method of preparing a micro or nano suspensioncomprising media components. This method may, for example, allow forconcentrating a medium and/or supplement way beyond solubility bymicrosuspension (ms) and/or nanosuspension (ns) of certain components,and further, by encapsulation of the microsuspension (ms) and/ornanosuspension (ns) to form beads, followed by desiccation of theencapsulated beads, further increase in concentration of the componentsoccur.

To make Microsuspensions (see FIG. 1)

For making microsuspensions of media components at ˜7× concentration(without sodium phosphate):

-   -   1) Weigh 30 g of dry format powdered media, supplement or feed,        without sodium phosphate, into a mortar.    -   2) Add 7.5 ml WFI (water for injection).    -   3) Using a green plastic pliable spatula, mix until the powder        is wetted and begins to “take up” the water and form a paste.        Thoroughly rapidly mix the paste for homogeneity, which is the        microsuspension.    -   4) Add 1 ml of additional WFI to the microsuspensions and mix        thoroughly.    -   5) Collect the microsuspensions into a container and use a        spatula to “squeegee” the last amount of microsuspensions into a        container for delivery. Volume will be ˜29 ml.    -   6) For delivery, use a piston-delivery device (for e.g.; a        syringe-type device).

Example 2: Microsuspensions and Nanosuspensions in AlginateMicrocapsules

Microencapsulation can provide a mechanism of physically separating orsequestering labile components, particularly from reactive components inpowdered cell culture media. By way of example, alginate or any otherencapsulating matrix or capsular material can be used formicroencapsulation.

Protocol for Making Encapsulated Microsuspensions

For making encapsulated microsuspensions, which is a way of furtherconcentrating media, supplement or feed:

-   -   1) Make a microsuspension of the media, supplement or feed        without sodium phosphate (for example, as described above).    -   2) Add 13.5 ml of 6% alginate into the microsuspension and mix        thoroughly with a spatula to blend in the alginate. Volume        should equal ˜40.5 ml.    -   3) Cut parafilm circles to cover the bottoms of 4-sided (medium)        polypropylene disposable weigh boats. Place parafilm into weigh        boat bottom.    -   4) Prepare Eppendorf Repeater-Plus tips by nipping off with        scizzors ˜ 1/64″ of end making sure that the cut is not as far        as the location of the end of plunger when fully depressed.    -   5) Using Eppendorf Repeater-Plus with 2.5 ml syringe set at        position 1 (25 μl), slowly pull microsuspension up into syringe.        Make sure that air is not aspirated into the syringe as this        would remain in the viscous microsuspension and alter volume        delivered.    -   6) Depress the Eppendorf lever several times to prime syringe.        After the microsuspension flows out of the syringe end, wipe off        the end of syringe. Then hold syringe end vertically a few mm        from parafilm surface, depress lever and keep at the same spot        for ˜5 seconds to allow all the 25 μl drop to be delivered to        the parafilm surface. Then move to an adjacent position and        repeat. Continue to place droplets over the entire surface of        the parafilm. The alginate-microsuspension droplet will form a        convex, spherical shape after sitting for a few seconds on the        parafilm.    -   7) To cross-link the alginate and form a hydrogel, deliver 25 ml        of a 133 g/L solution of calcium chloride (anhydrous) into the        weigh boat. It may be necessary to hold the parafilm (and        alginate-microsuspension droplets) underneath the calcium        chloride solution surface using tweezers since the droplet will        try to float, if the Ca solution gets underneath.    -   8) Hold the parafilm and alginate-microsuspension droplets under        the calcium chloride for 30 seconds. Forms a bead.    -   9) After 30 seconds, use tweezers to grab one end of the        parafilm and jostle to loosen and dislodge all the beads. It        helps to turn the parafilm upside down in the weigh boat and        swish back and forth in the calcium chloride. The beads will        readily come off the parafilm.    -   10) Pass through steps 11-18 without delay since        microsuspensions will be slowly dissolving throughout processing        until placed onto absorbent tissue paper (Step 16).    -   11) Immediately fold the weigh boat in half and pour off the        calcium chloride solution, being careful to hold the beads        within the weigh boat by the narrowed end.    -   12) Relax hold on the weigh boat and immediately add (pour)        enough WFI to half fill.    -   13) Immediately fold weigh boat as before and pour off WFI.    -   14) Relax hold on the weigh boat and immediately pour in enough        WFI to half fill.    -   15) Immediately fold weigh boat as before and pour off WFI.    -   16) Flip over and dump each weigh boat onto double tissue paper        to absorb as much water as possible from the beads.    -   17) Hold tissue paper over the weigh boat and scoop beads in        using plastic white flexible spatula.    -   18) Using 2 white plastic spatulas, separate beads so that they        do not touch each other.    -   19) Place under fume hood overnight.    -   20) Next morning, place beads into vacuum drying chamber over        CaSO₄.    -   21) Let dry for 3 days, then remove and dislodge beads from        surface of weigh boat where they will have a slight attachment.        Turn a clean weigh pan upside down and use to cover the beads in        their weigh pan. Lifting one end up slightly, push a white        flexible spatula across the surface of the weigh pan to dislodge        beads. After all are dislodged, place in the new weigh pan and        put back into vacuum drying chamber over CaSO₄ for 4 additional        days to assure dryness.

Example 3: Encapsulation of Minor Media Components Such as Vitamins

If the encapsulation targets are minor media components such asvitamins, Step I involves mixing a concentrate solution of the componentto be encapsulated directly into a 2% alginate solution. The solution isconcentrated enough so that it requires 1% or less of the volume of the2% alginate. No formation of microsuspensions are required. Thisalginate-component solution is then added as 25 μl drops fallingdirectly into 133 g/L CaCl2 solution and held for 30 seconds.

-   -   a) Drain beads of the CaCl2 solution and rinse rapidly 2×.    -   b) Separate the beads and dry on a flat surface for several days        to ensure moisture of <1%.    -   c) Then place in vacuum desiccators over CaSO4 for several days        until beads are refractile and hard.    -   d) Dry beads are ready for further use.

Using dried beads was like adding almost 100% nutrient supplementchemical to the bioreactor.

Example 4: Protocol for Applying Extended-Release Coating ontoAlginate-Encapsulated Microsuspensions

Since no access to bead coating equipment was available, a surrogateprocess was developed. It was found necessary to make sure absolutely noholes or weak areas would occur in the extended-release coating layer ofthe bead. Intact beads could not be repeatedly coated uniformly due tothe nature of the organic phase being added by hand. Therefore a slidesurrogate coating and testing assay was developed.

-   -   1) Make alginate-microsuspension as described above.    -   2) Using an Eppendorf pipettor, dot 5 μl of the        alginate-microsuspension within the clear glass circular areas        of the red ringed slide.    -   3) Immediately use a plastic spatula or other flexible tool to        “smoosh” or flatten the 5 μl alginate-microsuspension to fill as        much of the circular glass clear area as possible (goal is to        lower the surface of the alginate-microsuspension as low as        possible to assist the subsequent PLGA coating: tall beads do        NOT work as they project upwards too high on the slide).    -   4) Place slide into 133 g/L calcium chloride solution for ˜20-30        seconds.    -   5) Rinse by immersing in WFI.    -   6) Drain WFI off of slide (touch tissue to each well to suck up        excess water) and dry overnight in a fume hood.    -   7) Then place into vacuum over CaSO4 to desiccate.    -   8) PLGA stock at 25 mg/0.6 ml of chloroform. Add 50 μL PLGA in        chloroform to cover the well in the red ringed slide. The 50 μL        volume will “fill” the circular well area and bead up to give a        concave hump over the well. This volume of PLGA-chloroform will        readily evaporate to form a flat, contact-lens-like covering        over the dried alginate-microsuspension. Delay: 3 coats >2        coats>1 coat.    -   9) After the PLGA has dried, use to perform extended-release        comparisons with varying concentrations and glycolic to lactic        ratios.

Obtaining a uniform coating of a solution like PLGA onto a dried beadwas a challenge. Several attempts were made using a variety ofprotocols. The protocol that worked is presented above, which wasadapted from protocols till the desired coating level was obtained. Forinstance, flattening the bead provided the best coating results for theextended release use. Several parameters like coating componentconcentration, bead size, % alginate, protocols for coating, dryingconditions and level of dryness, solvents for pre-coating, etc. had tobe optimized in order to achieve the best results.

Example 5: Extended-Release Comparisons of Varying PLGA Concentrationsand Glycolic to Lactic Ratios: Assay

-   -   1. Place 55 ml of PBS into a 50 ml centrifuge tube.    -   2. Add one slide of the same PLGA composition that covers all        the 14 wells. Incubate horizontally at room temperature.    -   3. An exemplary encapsulated glucose bead was assay for glucose,        to study the extended-release properties of varying ratios of        the coating material components: glycolic to lactic ratios.    -   4. For sampling at various times, we performed the Sigma Glucose        Oxidase Assay (GAGO) for glucose:    -   a) 0.10 ml sample from the 50 ml centrifuge tube into a 10×75 mm        glass tube or a 1.5 ml Wheaton glass rubber stoppered chemical        analytical vial.    -   b) 0.20 ml of Reagent into the same tube, mix.    -   c) Incubate 15 min. at 37 C.    -   d) Add 0.20 ml 12N sulfuric acid, mix.    -   e) Controls: dilute a 1 g/L glucose solution 1:10 (0.1 ml+0.9 ml        WFI, equals 100 ug/ml); serial 2× dilutions (0.5 ml glucose+0.5        ml WFI) down to 12.5 ug/ml. Controls are 100, 50, 25 and 12.5        ug/ml glucose. Also include a WFI blank.    -   f) As a guide, when all of the microsuspension from all of the        14 wells is dissolved, sample needs to be diluted 1:4 to be        within range of control standards. [Total glucose applied should        be 0.022 g per slide at 14 wells, pg 69 NB 1138].    -   To read the output, use SpectraMax 384: 350 μl of samples in a        96 well tray. Either clear or dark walled work the same, no        difference. Read at 540 nm absorbance.    -   Calculations: [from Sigma Glucose (GO) Assay Kit, Product Code        GAGO-20]

mg Glucose=(ΔA@540 of Test) (mg Glucose in standard*)

-   -   * Concentration of closest standard of the 4 dilutions.    -   Δ means the difference between the OD reading of the test or        standard minus the reading of the blank.    -   **Multiply the mg Glucose determined above by the dilution        factor made in the sample preparation.

Example 6: Porous Metal Cylinder

A porous metal cylinder was filled with dried beads ofalginate-encapsulated microsuspensions and placed within a 1 L beakerwith a stir bar to simulate conditions inside a bioreactor. There was noPLGA coating of the beads for delayed-release. The graph in FIG. 11shows that stirring within the bioreactor alone is enough to get thenutrient supplements from the beads, and to dissolve and pass into the“bioreactor”. The porous metal cylinder needs no pumping, tubing, etc.to get the supplement to go into the cell culture. In fact, it is sofast that in one test experiment, the supplement in alginate beads alone(no PLGA coating) completely liberates its load within about 20 hours.Extended-release requires PLGA coating even when beads are within theporous metal. The lower chart of FIG. 11 shows that AGT powder alone canpass through the porous metal cylinder relatively quickly, but AGT alonewould not work for delayed-release. A point to note is that the porousmetal cylinder can be autoclaved attached to the bioreactor, and thesupplement beads can be added at anytime during the culture.

FIG. 11 shows two ways that extended-release microcapsules could be usedfor nutrient supplementation of bioreactors. 1) Sterile microcapsulescould be added directly to the bioreactor where they release componentsin a time-release manner (right), or 2) they may be added to a porousmetal cylinder with 0.22μ pore size, keeping the beads away fromcontacting the cells in culture. With option 1, the beads may interferewith filtration, or may reduce cell count or productivity. With option2, bioreactor stirring alone is enough to dissolve nutrient supplementfrom inside the porous metal cage and to pass into the bioreactor.

Some Exemplary Embodiments

A method of making a media, feed or supplement composition, the methodcomprising:

-   -   adding a minimal volume of an aqueous solution to a dry powder        of the media, feed or supplement to make a paste;    -   mixing the paste vigorously to prepare a microsuspension.

A method of preparing a media composition comprising:

-   -   preparing a microsuspension of the media according to claim 1;    -   optionally, mixing in an effective amount of an anti-oxidant        with the microsuspension to form a mixture;    -   encapsulating the microsuspension, or the mixture of step 2, in        a suitable capsular material such that a microcapsule or bead is        formed;    -   drying the microcapsule or bead,    -   wherein the media comprises a labile substance.

The method of claim 2, wherein the labile substance is attached to adendrimer.

The method of claim 2, wherein the media is prepared for one or more ofthe properties selected from the group consisting of: at least onecomponent is in super concentration, for increased stability, forincreased resistance to radiation exposure, for thermostability, forextended shelf-life and for extended release of the labile component.

The method of claim 2, wherein the media composition is assayed forassays selected from the group consisting of assay for extended-releaseof encapsulated components, assay for thermostability, assay forreduction of viral numbers, assay for functionality after irradiation,assay for extended shelf-life, assay for stability during transport andassay for storage at ambient temperatures.

The method of claim 2, wherein the capsular material is selected fromthe group consisting of alginate, poly-L-lactic acid, chitosan, agarose,gelatin, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate,heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guargum, water soluble cellulose derivatives and carrageenan.

The method of claim 2, wherein the bead if further coated with a coatingthat extends the release of the labile component from the bead.

The method of claim 5, wherein the coating is selected from the groupconsisting of poly-glycolic acid, PLGA (poly-lactic-co-glycolic acid),collagen, polyhydroxy-alkanoates (PHA), poly-ε-caprolactone, poly-orthoesters, poly-anhydrides, poly-phosphazenes, poly-amino acids,polydimethylsiloxane, polyurethranes, poly-tetrafluoroethylene,polyethylene, polysulphone, poly-methyl methacrylate,poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinylchloride, polystyrene and poly-vinyl pyrrolidone.

The method of claim 2, wherein the bead is irradiated.

The method of claim 8, wherein the bead is assayed for sterility.

The method of claim 4, wherein the bead is irradiated withgamma-irradiation.

The method of claim 5, wherein the bead is additionally irradiated withUV rays.

The method of claim 5, wherein the bead is free of PPV and MMV viruses.

The method of claim 1 or 2, wherein the composition is a dry-formatmedia.

The method of claim 1 or 2, wherein the labile component is selectedfrom the group consisting of a polyamine, a growth factor, a cytokineand a vitamin

The method of claim 2, wherein the capsular material is soluble uponreconstitution with an aqueous solvent.

The method of claim 3, wherein the encapsulating matrix encapsulates adendrimer-labile component complex.

The method of claim 14, wherein the dendrimer is a polyamidoaminedendrimer, a polypropylenimine dendrimer, or a polypropylamine (POPAM)dendrimer.

The method of any one of claims 1-15, wherein the composition comprisesa powdered cell culture medium comprising one or more amino acids.

A medium, feed or supplement composition comprising a microsuspensioncomprising a component.

A medium, feed or supplement composition comprising a mixture of alabile component and an anti-oxidant that is microencapsulated with acapsular matrix into a bead.

The medium, feed or supplement composition of claim 18, wherein thecapsular material that is selected from the group consisting ofalginate, poly-L-lactic acid, chitosan, agarose, gelatin, hyaluronicacid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparinsulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, watersoluble cellulose derivatives and carrageenan.

The medium, feed or supplement composition of claim 18, wherein the beadis coated with a coating solution.

The medium, feed or supplement composition of claim 20, wherein thecoating solution is selected form the group consisting of poly-glycolicacid, PLGA (poly-lactic-co-glycolic acid), collagen,polyhydroxyalkanoates (PHA), poly-ε-caprolactone, poly-ortho esters,poly-anhydrides, poly-phosphazenes, poly-amino acids,polydimethylsiloxane, polyurethranes, poly-tetrafluoroethylene,polyethylene, polysulphone, poly-methyl methacrylate,poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinylchloride, polystyrene, poly-vinyl pyrrolidone, poly-L-lysine andpolyornithine.

The medium, feed or supplement composition of claim 18, wherein thecomposition further comprises chelated reactive species.

The medium, feed or supplement composition of claim 21, wherein thereactive species are either cations, metals ions or trace elements.

The medium, feed or supplement composition of claim 21, wherein thechelating moieties are selected from the group consisting of EDTA,citrate, succinate, cyclodextrin, clatharates, dendrimers and aminoacids.

The medium, feed or supplement composition of any of claims 17-23,wherein the composition is irradiated.

The medium, feed or supplement composition of claim 24 wherein theirradiation is with gamma-rays.

The medium, feed or supplement composition of claim 25, wherein thegamma-rays are 25-100 kGy.

The medium, feed or supplement composition of claim 26, wherein thegamma-rays are 30-50 kGy.

The medium, feed or supplement composition of claim 26, wherein thegamma-ray is 30 kGy.

The medium, feed or supplement composition of any of 17-28 which isadded aseptically into a bioreactor.

The medium, feed or supplement composition of any of 17-29, wherein thecell culture medium, feed or supplement is protein free.

The medium, feed or supplement composition of any of 17-30, wherein thepowdered cell culture medium used for the microsuspension is AGT(advanced granulation technology cell culture medium).

A use of any of the compositions described above to produce a dry formatcell culture medium.

A use of any of the compositions described above to increase the shelflife of the cell culture medium.

A use of any of the compositions described above to store and handle atambient temperatures.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present specification, includingdefinitions, will control. It will be readily apparent to one ofordinary skill in the relevant arts that other suitable modificationsand adaptations to the methods and applications described herein areobvious and may be made without departing from the scope of thedisclosure or any embodiment thereof. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting. All patents, patent applications, and published referencescited herein are hereby incorporated by reference in their entirety.

What is claimed is:
 1. A process for extending the release of acomponent in a cell culture medium, feed or supplement, the processcomprising: (i) adding a minimal volume of an aqueous solution to a drypowder cell culture medium, feed or supplement comprising the componentfor extended release, to make a paste; (ii) mixing the paste vigorouslyto prepare a microsuspension; (iii) optionally, adding an effectiveamount of an anti-oxidant to the microsuspension of (ii) to form amixture; (iv) encapsulating the microsuspension of (ii), or the mixtureof (iii), into a capsular material to form a bead; and (v) drying thebead.
 2. The process of claim 1, wherein the component is selected fromthe group consisting of glucose, a vitamin, an amino acid or itsderivative, a peptides, a macromolecule, a polyamine, a hormone, agrowth factor and a labile substance.
 3. The process of claim 2, whereinthe microsuspension of step (iv) concentrates the contents within thebead.
 4. The process of claim 1, wherein the capsular material isselected from the group consisting of alginate, poly-L-lactic acid,chitosan, agarose, gelatin, hyaluronic acid, chondroitin sulfate,dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate,gellan gum, xanthan gum, guar gum, water soluble cellulose derivativesand carrageenan.
 5. The process of claim 1, wherein the bead is coatedwith a coating solution that further extends the release of thecomponent from the bead.
 6. The process of claim 5, wherein the coatingsolution is selected form the group consisting of poly-glycolic acid,PLGA (poly-lactic-co-glycolic acid), collagen, polyhydroxyalkanoates(PHA), poly-ε-caprolactone, poly-ortho esters, poly-anhydrides,poly-phosphazenes, poly-amino acids, polydimethylsiloxane,polyurethranes, poly-tetrafluoroethylene, polyethylene, polysulphone,poly-methyl methacrylate, poly-2-hydroxyethylmethacrylate, polyamides,polypropylene, poly-vinyl chloride, polystyrene, poly-vinyl pyrrolidone,poly-L-lysine and polyornithine.
 7. The process of claim 6, whereingreater than 50% of the extended release component in themicrosuspension is present after 15 days in culture.
 8. The process ofclaim 4, wherein the capsular material encapsulates adendrimer-component complex.
 9. The process of claim 8, wherein thedendrimer is a polyamidoamine dendrimer, a polypropylenimine dendrimer,or a polypropylamine (POPAM) dendrimer.
 10. The process of claim 1,wherein dry powder cell culture medium, feed or supplement is eitherfine-milled or AGT (advanced granulation technology cell culturemedium).
 11. The process of claim 1, wherein the composition furthercomprises chelated reactive species.
 12. The process of claim 11,wherein the reactive species is a cation, a metal ion, or a traceelement.
 13. The process of claim 11, wherein the chelating moieties areselected from the group consisting of EDTA, citrate, succinate,cyclodextrin, clatharates, dendrimers and amino acids.
 14. The processof claim 1, wherein the cell culture medium, feed or supplement isirradiated.
 15. The process of claim 14, wherein the cell culturemedium, feed or supplement is free of PPV and MMV viruses.
 16. Use ofthe cell culture medium, feed or supplement comprising the extendedrelease component of claim 2, to produce a liquid medium for culturing acell. wherein the delayed-release of the component into the cell culturefor over 6-24 hours, 24-48 hours, 48-72 hours, over 1 day, 2 days, 3-5days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-40days, 40-50 days, over the duration of the culture.
 17. The use of thecell culture medium, feed or supplement of claim 16, wherein the medium,feed or supplement comprising the extended release component is storedat ambient temperatures.
 18. The use of the cell culture medium, feed orsupplement of claim 1 wherein the cell is a mammalian cell.
 19. The useof the cell culture medium, feed or supplement of claim 18, wherein themammalian cell is selected from the group consisting of Chinese HamsterOvary (CHO) cells, 293, BHK, Vero, PerC6, MDBK and MDCK cells.
 20. Theuse of the cell culture medium, feed or supplement of claim 18, whereinthe mammalian cell is a CHO cell.